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| Status |
Public on Mar 21, 2017 |
| Title |
Transcriptome mapping and Hfq RIP-seq of Neisseria meningitidis |
| Organism |
Neisseria meningitidis 8013 |
| Experiment type |
Expression profiling by high throughput sequencing
Other
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| Summary |
Neisseria meningitidis is a human commensal that occasionally causes life-threatening infections such as bacterial meningitis and septicemia. Despite experimental evidence that gene regulation as well as the expression of small non-coding RNAs (sRNAs) affect meningococcal virulence, the organization of its transcriptome, including in particular the biogenesis of sRNAs and their mode of action, is only poorly understood. Here, we addressed these issues using a combination of high-throughput technologies. We applied differential RNA-seq (dRNA-seq) to produce a single-nucleotide resolution map of the primary transcriptome of N. meningitidis strain 8103. Our dRNA-seq analysis predicted 1,625 transcriptional start sites (TSS) including 65 non-coding RNA transcripts, of which 20 were further validated by Northern analysis. This allowed for the discovery of a novel CRISPR-associated sRNA with a Cas9-independent biogenesis. Genome-wide mapping of σ 70-dependent and independent promoters revealed that classical Escherichia coli-like σ70 promoter are absent in most of the protein coding genes in meningococci. The majority of the 706 primary TSSs (pTSSs) were associated with coding sequences, including 382 pTSS obtained for single genes and 240 pTSSs obtained for genes located in operons. By Hfq RNA immunoprecipitation sequencing (RIP-seq) we identified a large Hfq-centered post-transcriptional regulatory network comprising 24 sRNAs and 407 potential mRNA targets, and rifampicin stability assays demonstrated that Hfq binding confers enhanced stability on sRNAs. We finally confirmed the interactions of two sRNAs and their cognate target mRNA in vivo. Both directly repress prpB encoding a methylisocitrate lyse which was previously shown to be involved in meningococcal colonization of the human nasopharynx.The combination of both high-throughput approaches thus creates a compendium that not only provides a valuable data resource, but also allows for a better understanding of meningococcal transcriptome organization and riboregulation with implications for colonization of the human nasopharynx.
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| |
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| Overall design |
differential RNA-seq at two different growth phases; Hfq RIP-seq
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| |
|
| Contributor(s) |
Barquist L, Heidrich N |
| Citation(s) |
28334889 |
| |
| Submission date |
Aug 05, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Lars Barquist |
| E-mail(s) |
[email protected]
|
| Organization name |
Helmholtz Institute for RNA-based Infection Research
|
| Street address |
Josef-Schneider-Straße 2 / Bau D15
|
| City |
Würzburg |
| State/province |
Bayern |
| ZIP/Postal code |
97080 |
| Country |
Germany |
| |
|
| Platforms (2) |
| GPL22295 |
Illumina HiSeq 2000 (Neisseria meningitidis 8013) |
| GPL22296 |
Illumina MiSeq (Neisseria meningitidis 8013) |
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| Samples (8)
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| Relations |
| BioProject |
PRJNA337954 |
| SRA |
SRP081012 |
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